Beta-amino acid derivatives as integrin receptor antagonists

ABSTRACT

The present invention relates to compounds of the general formula (I), processes for their preparation, pharmaceutical compositions containing them as well as their use for the production of pharmaceutical compositions for the treatment of inflammatory diseases.

[0001] The present invention relates to compounds of formula (I),

[0002] their preparation and use as pharmaceutical compositions asintegrin antagonists, especially as α₄β₁ and/or α₄β₇ and/or α₉β₁integrin antagonists and in particular for the production ofpharmaceutical compositions suitable for the inhibition or theprevention of cell adhesion and cell-adhesion mediated disorders.

[0003] Adhesive interactions between the leukocytes and endothelialcells play a critical role in leukocyte trafficking to sites ofinflammation. These events are essential for normal host defense againstpathogens and repair of tissue damage, but can also contribute to thepathology of a variety of inflammatory and autoimmune disorders. Indeed,eosinophil and T cell infiltration into the tissue is known as acardinal feature of allergic inflammation such as asthma.

[0004] The interaction of circulating leukocytes with adhesion moleculeson the luminal surface of blood vessels appears to modulate leukocytetransmigration. These vascular cell adhesion molecules arrestcirculating leukocytes, thereby serving as the first step in theirrecruitment to infected or inflamed tissue sites. Subsequently, theleukocytes reaching the extravascular space interact with connectivetissue cells such as fibroblasts as well as extracellular matrixproteins such as fibronectin, laminin, and collagen. Adhesion moleculeson the leukocytes and on the vascular endothelium are hence essential toleukocyte migration and attractive therapeutic targets for interventionin many inflammatory disorders.

[0005] Leukocyte recruitment to sites of inflammation occurs in astepwise fashion beginning with leukocyte tethering to the endothelialcells lining the blood vessels. This is followed by leukocyte rolling,activation, firm adhesion, and transmigration. A number of cell adhesionmolecules involved in those four recruitment steps have been identifiedand characterized to date. Among them, the interaction between vascularcell adhesion molecule 1 (VCAM-1) and very late antigen 4 (VLA-4, α₄β₁integrin), as well as the interaction between mucosal addressin celladhesion molecule 1 (MAdCAM-1) and α₄β₇ integrin, has been shown tomediate the tethering, rolling, and adhesion of lymphocytes andeosinophils, but not neutrophils, to endothelial cells under aphysiologic flow condition. This suggests that the VCAM-1/VLA-4 and/orMAdCAM-1/α₄β₇ integrin mediated interactions could predominantly mediatea selective recruitment of leukocyte subpopulations in vivo. Theinhibition of this interaction is a point of departure for therapeuticintervention (A. J. Wardlaw, J. Allergy Clin. Immunol. 1999, 104,917-26).

[0006] VCAM-1 is a member of immunoglobulin (Ig) superfamily and is oneof the key regulators of leukocyte trafficking to sites of inflammation.VCAM-1, along with intracellular adhesion molecule 1 (ICAM-1) andE-selectin, is expressed on inflamed endothelium activated by suchcytokines as interleukin 1 (IL-1) and tumor necrosis factor α (TNF-α),as well as by lipopolysaccharide (LPS), via nuclear factor κB (NF-κB)dependent pathway. However, these molecules are not expressed on restingendothelium. Cell adhesion mediated by VCAM-1 may be involved innumerous physiological and pathological processes including myogenesis,hematopoiesis, inflammatory reactions, and the development of autoimmunedisorders. Integrins VLA-4 and α₄β₇ both function as leukocyte receptorsfor VCAM-1.

[0007] The integrin α₄β₁ is a heterodimeric protein expressed insubstantial levels on all circulating leukocytes except matureneutrophils. It regulates cell migration into tissues duringinflammatory responses and normal lymphocyte trafficking. VLA-4 binds todifferent primary sequence determinants, such as a QIDSP motif of VCAM-1and an ILDVP sequence of the major cell type-specific adhesion site ofthe alternatively spliced type III connecting segment domain (CS-1) offibronectin.

[0008] In vivo studies with neutralizing monoclonal antibodies andinhibitor peptides have demonstrated a critical role for α₄ integrinsinteraction in leukocyte-mediated inflammation. Blocking of VLA-4/ligandinteractions, thus, holds promise for therapeutic intervention in avariety of inflammatory, autoimmune and immune diseases (Zimmerman, C.;Exp. Opin. Ther. Patents 1999, 9, 129-133).

[0009] Furthermore, compounds containing a bisarylurea moiety as asubstituent were disclosed as α₄β₁ integrin receptor antagonists: WO96/22966, WO 97/03094, WO99/20272, WO99/26923, WO 99/33789, WO 99/37605,WO 00/00477. However, no β-amino acids or homologues thereof with α₄β₁integrin receptor antagonistic activity have been described.

[0010] Further to their α₄β₁ integrin antagonistic activity, thecompounds of the present invention may also be used as α₄β₇ or α₉β₁integrin antagonists.

[0011] An object of the present invention is to provide new β-amino acidor homologues thereof derived integrin antagonists for the treatment ofinflammatory, autoimmune and immune diseases.

[0012] The present invention therefore relates to compounds of thegeneral formula (I):

[0013] wherein

[0014] R¹ represents a 4-to 9-membered saturated, unsaturated oraromatic cyclic residue,

[0015] which can contain 0 to 3 heteroatoms selected independently fromthe group N, S and 0,

[0016] and wherein R¹ is substituted by —R¹⁻¹—Z,

[0017] wherein

[0018] R¹⁻¹ represents a bond, —O—, —S—, NR¹⁻², C₁-C₁₀ alkyl, C2-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₆ or C₁₀ aryl, C₃-C₇ cycloalkyl or a4-9-membered saturated or unsaturated heterocyclic residue containing upto 3 heteroatoms selected from the group oxygen, nitrogen or sulfur,

[0019] wherein R¹⁻¹ can optionally be substituted by 1 to 2 substituentsselected from the group R¹⁻³,

[0020] wherein R¹⁻² can optionally be hydrogen, C₁-C₁₀ alkyl, C₂-C₁₀alkenyl or C₂-C₁₀ alkynyl, and

[0021] wherein R¹⁻³ represents hydrogen, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl,C₂-C₁₀ alkynyl, C₆ or C₁₀ aryl, C₃-C₇ cycloalkyl or a 4-9-memberedsaturated or unsaturated heterocyclic residue containing up to 3heteroatoms selected from the group oxygen, nitrogen or sulfur,

[0022] Z represents —C(O)OR^(Z-1), —C(O)NR^(Z-2)R^(Z-3),—SO₂NR^(Z-2)R^(Z-3), —SO(OR^(Z-1)), —SO₂(OR^(Z-1)),—P(O)R^(Z-1)(OR^(Z-3)) or —PO(OR^(Z-1))(OR^(Z-3)),

[0023] wherein R^(Z-2) is hydrogen, C₁-C₄ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₃-C₆ cycloalkyl, C₆ or C₁₀ aryl, —C(O)R^(Z-4) or —SO₂R^(Z-4),

[0024] wherein R^(Z-4) is C₁-C₄ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,C₃-C₆ cycloalkyl, C₆ or C₁₀ aryl,

[0025] R^(Z-1) and R^(Z-3) are independently selected from the grouphydrogen, C₁-C₄ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl,C₆ or C₁₀ aryl or benzyl,

[0026] wherein R^(Z-1) and R^(Z-3) can optionally be substituted by 1 to3 substituents selected from the group C₁-C₄ alkyl, C₁-C₄ alkyloxy,halogen, nitro, cyano,

[0027] and wherein R¹ can optionally be substituted by 0 to 2substituents R¹⁻⁴, halogen, nitro, amino, cyano and oxo,

[0028] wherein

[0029] R¹⁻⁴ is selected from the group C₁-C₄ alkyl, C₁-C₄ alkyloxy,phenyl, phenoxy, phenylamino, C₃-C₆ cycloalkyl,

[0030] R² represents hydrogen, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀alkynyl, C₆ or C₁₀ aryl or C₃-C₇ cycloalkyl,

[0031] wherein R² can optionally be substituted by 1 to 3 radicalsindependently selected from the group C₁-C₄ alkyl, trifluormethyl,trifluormethoxy, halogen, cyano, nitro or oxo,

[0032] R³ represents hydrogen, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀alkynyl, C₆ or C₁₀ aryl, C₃-C₇ cycloalkyl or a 4-9-membered saturated orunsaturated heterocyclic residue containing up to 2 heteroatoms selectedfrom the group oxygen, nitrogen or sulfur,

[0033] wherein R³ can optionally be substituted by 1 to 3 radicals R³⁻¹,

[0034] wherein R³⁻¹ represents C₁-C₄ alkyl, trifluoroethyl,trifluormethoxy, —OR³⁻², —SR³⁻², NR³⁻³R³⁻⁴, —C(O)R³⁻², S(O)R³⁻²,—SO₂R³⁻², —OC(O)R³⁻², —C(O)NR³⁻³R³⁻⁴, —NR³⁻²C(O)R³⁻³, —SO₂NR³⁻³R³⁻⁴,NR³⁻²SO₂R³⁻³, —NR³⁻²C(O)NR³⁻³R³⁻⁴, —NR³⁻²C(O)OR³⁻³, —OC(O)NR³⁻³R³⁻⁴,—CO₂R³⁻⁵, halogen, cyano, nitro or oxo,

[0035] wherein R³⁻² represents hydrogen, C₁-C₄ alkyl, C₃-C₆ cycloalkyl,C₆ or C₁₀ aryl,

[0036] wherein R³⁻³ and R³⁻⁴ are independently selected from the grouphydrogen, C₁-C₄ alkyl, C₃-C₆ cycloalkyl, C₆ or C₁₀ aryl or benzyl,

[0037] and wherein R³⁻⁵ represents C₁-C₄ alkyl, C₃-C₆ cycloalkyl, C₆ orC₁₀ aryl,

[0038] R⁴ represents hydrogen, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀alkynyl, C₆ or C₁₀ aryl, C₃-C₇ cycloalkyl or a 4-9-membered saturated orunsaturated heterocyclic residue containing up to 2 heteroatoms selectedfrom the group oxygen, nitrogen or sulfur,

[0039] wherein R⁴ can optionally be substituted by 1 to 3 radicals R⁴⁻¹,

[0040] wherein R⁴⁻¹ represents C₁-C₄ alkyl, trifluormethyl,trifluormethoxy, —OR⁴⁻², —SR⁴⁻², NR⁴⁻³R⁴⁻⁴, —C(O)R⁴⁻², S(O)R⁴⁻²,—SO₂R⁴⁻², —OC(O)R⁴⁻², —C(O)NR⁴⁻³R⁴⁻⁴, —NR⁴⁻²C(O)R⁴⁻³, —SO₂NR⁴⁻³R⁴⁻⁴,NR⁴⁻²SO₂R⁴⁻³, —NR⁴⁻²C(O)NR⁴⁻³R⁴⁻⁴, —NR⁴⁻²C(O)OR⁴⁻³, —OC(O)NR⁴⁻³R⁴⁻⁴,—CO₂R⁴⁻⁵, halogen, cyano, nitro or oxo,

[0041] wherein R⁴⁻² represents hydrogen, C₁-C₄ alkyl, C₃-C₆ cycloalkyl,C₆ or C₁₀ aryl,

[0042] wherein R⁴⁻³ and R⁴⁻⁴ are independently selected from the grouphydrogen, C₁-C₄ alkyl, C₃-C₆ cycloalkyl, C₆ or C₁₀ aryl or benzyl,

[0043] and wherein R⁴⁻⁵ represents C₁-C₄ alkyl, C₃-C₆ cycloalkyl, C₆ orC₁₀ aryl

[0044] or

[0045] R³ and R⁴ together with the carbon atom to which they areattached form a 4-7-membered saturated or unsaturated ring containing upto 2 heteroatoms selected from the group oxygen, nitrogen or sulfur,which can optionally be substituted by 1 to 2 substituents selected fromthe group C₁-C₄ alkyl, phenyl, benzyl, C₃-C₇ cycloalkyl, C₁-C₄ alkyloxy,halogen, nitro, cyano, oxo and which can be fused with a 3-7 memberedhomocyclic or heterocyclic, saturated, unsaturated or aromatic ring,

[0046] R⁵ represents hydrogen, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀alkynyl, C₆ or C₁₀ aryl or C₃-C₇ cycloalkyl,

[0047] wherein R⁵ can optionally up to threefoldedly be substituted byC₁-C₄ alkyl, trifluormethyl, trifluormethoxy, halogen, cyano, nitro oroxo,

[0048] R⁶ represents phenyl or a 5- to 6-membered aromatic heterocyclicresidue containing up to 3 heteroatoms independently selected from thegroup oxygen, nitrogen or sulfur,

[0049] which is substituted by —NR⁶⁻²C(O)NR⁶⁻³R⁶⁻⁴ or—NR⁶⁻²C(S)NR⁶⁻³R⁶⁻⁴ and can furthermore optionally be substituted byhalogen,

[0050] wherein R⁶⁻² and R⁶⁻³ are independently selected from the grouphydrogen or C₁-C₄ alkyl, or together form a group

[0051] and wherein R⁶⁻⁴ represents phenyl,

[0052] wherein R⁶⁻⁴ can optionally be substituted by 1-2 substituentsselected from the group C₁-C₄ alkyl, C₁-C₄ alkyloxy, halogen, nitro,trifluoromethyl, trifluoromethoxy or cyano,

[0053] R⁷ represents hydrogen, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀alkynyl, C₆ or C₁₀ aryl, C₃-C₇ cycloalkyl or a 4-9-membered saturated orunsaturated heterocyclic residue containing up to 2 heteroatoms selectedfrom the group oxygen, nitrogen or sulfur,

[0054] wherein R⁷ can optionally be substituted by 1 to 3 radicals R⁷⁻¹,

[0055] wherein R⁷⁻¹ represents C₁-C₄ alkyl, trifluormethyl,trifluormnethoxy, —OR⁷⁻², —SR⁷⁻², NR⁷⁻³R⁷⁻⁴, —C(O)R⁷⁻², S(O)R⁷⁻²,—SO₂R⁷⁻², —OC(O)R⁷⁻², —C(O)NR⁷⁻³R⁷⁻⁴, —NR⁷⁻²C(O)R⁷⁻³, —SO₂NR⁷⁻³R⁷⁻⁴,NR⁷⁻²SO₂R⁷⁻³, —NR⁷⁻²C(O)NR⁷⁻³R⁷⁻⁴, —NR⁷⁻²C(O)OR⁷⁻³, —OC(O)NR⁷⁻³R⁷⁻⁴,—CO₂R⁷⁻⁵, halogen, cyano, nitro or oxo,

[0056] wherein R⁷⁻² represents hydrogen, C₁-C₄ alkyl, C₃-C₆ cycloalkyl,C₆ or C₁₀ aryl,

[0057] wherein R⁷⁻³ and R⁷⁻⁴ are independently selected from the grouphydrogen, C₁-C₄ alkyl, C₃-C₆ cycloalkyl, C₆ or C₁₀ aryl or benzyl,

[0058] and wherein R⁷⁻⁵ represents C₁-C₄ alkyl, C₃-C₆ cycloalkyl, C₆ orC₁₀ aryl

[0059] R⁸ represents hydrogen, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀alkynyl, C₆ or C₁₀ aryl, C₃-C₇ cycloalkyl or a 4-9-membered saturated orunsaturated heterocyclic residue containing up to 2 heteroatoms selectedfrom the group oxygen, nitrogen or sulfur,

[0060] wherein R⁸ can optionally be substituted by 1 to 3 radicals R⁸⁻¹,

[0061] wherein R⁸⁻¹ represents C₁-C₄ alkyl, trifluorrnethyl,trifluormethoxy, —OR⁸⁻², —SR⁸⁻², NR⁸⁻³R⁸⁻⁴, —C(O)R⁸⁻², S(O)R⁸⁻²,—SO₂R⁸⁻², —OC(O)R⁸⁻², —C(O)NR⁸⁻³R⁸⁻⁴, —NR⁸⁻²C(O)R⁸⁻³, —SO₂NR⁸⁻³R⁸⁻⁴,NR⁸⁻²SO₂R⁸⁻³, —NR⁸⁻²C(O)NR⁸⁻³R⁸⁻⁴, —NR⁸⁻²C(O)OR⁸⁻³, —OC(O)NR⁸⁻³R⁸⁻⁴,—CO₂R⁸⁻⁵, halogen, cyano, nitro or oxo,

[0062] wherein R⁸⁻² represents hydrogen, C₁-C₄ alkyl, C₃-C₆ cycloalkyl,C₆ or C₁₀ aryl,

[0063] wherein R⁸⁻³ and R⁸⁻⁴ are independently selected from the grouphydrogen, C₁-C₄ alkyl, C₃-C₆ cycloalkyl, C₆ or C₁₀ aryl or benzyl,

[0064] and wherein R⁸⁻⁵ represents C₁-C₄ alkyl, C₃-C₆ cycloalkyl, C₆ orC₁₀ aryl

[0065] or

[0066] R⁷ and R⁸ together form a 4-7-membered saturated or unsaturatedring containing up to 2 heteroatoms selected from the group oxygen,nitrogen or sulfur, which can optionally be substituted by 1 to 2substituents selected from the group C₁-C₄ alkyl, phenyl, benzyl, C₃-C₇cycloalkyl, C₁-C₄ alkyloxy, halogen, nitro, cyano, oxo and which can befused with a 3-7 membered homocyclic or heterocyclic, saturated,unsaturated or aromatic ring,

[0067] X represents bond or (—CR^(X-1)R^(X-2))_(n),

[0068] wherein R^(X-1) and R^(X-2) can be independently selected fromthe group hydrogen, C₁-C₄ alkyl, C₂-C₄ alkenyl, C₂-C₄ alkynyl,

[0069] wherein R^(X-1) and R^(X-2) can optionally independently besubstituted by 1 to 2 substituents selected from the group C₁-C₄ alkyl,phenyl, benzyl, C₃-C₇ cycloalkyl, C₁-C₄ alkyloxy, halogen, nitro, cyano,oxo,

[0070] and wherein n is an integer 0 or 1,

[0071] and pharmaceutically acceptable salts thereof.

[0072] In the context of the present invention alkyl stands for astraight-chain or branched alkyl residue, such as methyl, ethyl,n-propyl, iso-propyl, n-pentyl. If not stated otherwise, preferred isC₁-C₁₀ alkyl, very preferred is C₁-C₆ alkyl.

[0073] Alkenyl and alkinyl stand for straight-chain or branched residuescontaining one or more double or triple bonds, e.g. vinyl, allyl,isopropinyl, ethinyl. If not stated otherwise, preferred is C₁-C₁₀alkenyl or alkinyl, very preferred is C₁-C₆ alkenyl or alkinyl.

[0074] Cycloalkyl stands for a cyclic alkyl group such as cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl. Preferred ismonocyclic C₃-C₇ cycloalkyl.

[0075] Halogen in the context of the present invention stands forfluorine, chlorine, bromine or iodine. If not specified otherwise,chlorine or fluorine are preferred.

[0076] Homocycle stands for a ring consisting of carbon atoms.

[0077] A 4-to 9-membered saturated, unsaturated or aromatic cyclicresidue stands for a monocyclic system containing 4 to 9 ring atoms andcontaining 0, 1 or more double bonds, which can be attached via a carbonatom or eventually via a heteroatom within the ring, for example phenyl,thiazolyl, pyridyl, cyclopentyl.

[0078] Aryl stands for a monocyclic Hueckel-aromatic cyclic systemcontaining 6 or 10 ring carbon atoms.

[0079] Heteroaryl stands for a monocyclic heteroaromatic systemcontaining 4 to 9 ring atoms, which can be attached via a carbon atom oreventually via a nitrogen atom within the ring, for example, furan-2-yl,furan-3-yl, pyrrol-1-yl, pyrrol-2-yl, pyrrol-3-yl, thienyl, thiazolyl,oxazolyl, imidazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidyl orpyridazinyl.

[0080] A saturated or unsaturated heterocyclic residue stands for aheterocyclic system containing 4 to 9 ring atoms, which can contain oneor more double bonds and which can be attached via a ring carbon atom oreventually via a nitrogen atom, e.g. tetrahydrofur-2-yl,pyrrolidine-1-yl, piperidine-1-yl, piperidine-2-yl, piperidine-3-yl,piperidine-4-yl, piperazine-1-yl, piperazine-2-yl morpholine-1-yl,1,4-diazepine-1-yl or 1,4-dihydropyridine-1-yl.

[0081] If not specified otherwise, in the context of the presentinvention heteroatom stands preferably for O, S, N or P.

[0082] In a preferred embodiment, the present invention relates tocompounds of general formula (I), wherein R¹ represents a phenyl ring.

[0083] In another preferred embodiment, the present invention relates tocompounds of general formula (I), wherein R¹⁻¹ represents a bond and Zrepresents COOR^(Z-1), wherein R^(Z-1) has the meaning indicated above.

[0084] In yet another preferred embodiment, the present inventionrelates to compounds of general formula (I), wherein R⁶ representsphenyl, which is substituted by —NHC(O)NHR⁶⁻⁴, wherein R⁶⁻⁴ issubstituted with methyl or trifluoromethoxy.

[0085] In yet another preferred embodiment, the present inventionrelates to compounds of general formula (I), wherein R^(X-1) and R^(X-2)represent hydrogen.

[0086] In yet another preferred embodiment, the present inventionrelates to compounds of general formula (I), wherein R³ and R⁴ togetherform a 6-membered homocycle.

[0087] In yet another preferred embodiment, the present inventionrelates to compounds of general formula (I), wherein R³, R⁴ and R⁷represent hydrogen and R⁸ represents a 3-methoxyphenylradical or a3,4-dimethoxyphenylradical.

[0088] In yet another preferred embodiment, the present inventionrelates to compounds of general formula (I), wherein R¹ is a1,4-substituted phenyl ring.

[0089] Particularily preferred are the following compounds:

[0090]4-{[(1-(3,4-Dimethoxyphenyl)-3-{[4-({[(2-methylphenyl)amino]carbonyl}amino)phenyl]amino}-3-oxopropyl)amino]carbonyl}benzoicacid

[0091]4-[({[1-({[4-({[(2-Methylphenyl)amino]carbonyl}amino)benzyl]amino}carbonyl)cyclohexyl]methyl}amino)carbonyl]benzoicacid

[0092]4-{[(1-(3,4-Dimethoxyphenyl)-3-{[4-({[(2-methylphenyl)amino]carbonyl}amino)benzyl]amino}-3-oxopropyl)amino]caronyl}benzoicacid

[0093]4-{[(1-(3-Methoxyphenyl)-3-{[4-({[(2-methylphenyl)amino]carbonyl}amino)phenyl]amino}-3-oxopropyl)amino]caronyl}benzoicacid

[0094] and their respective tert-butyl-benzoates.

[0095] A preferred process for preparation of compounds of generalformula (I) has also been found, which comprises reaction of carboxylicacids of general formula (I′)

[0096] or activated derivatives thereof,

[0097] with compounds of the general formula (I″)

R⁶—X—NR⁵H  (I″)

[0098] in inert solvents, which will be described in more detail in thedescriptive part of the specification.

[0099] The intermediates (I′), wherein R¹, R², R³, R⁴, R⁷ and R⁸ havethe abovementioned meaning and PG₁ represents a protecting group for thecarboxyl group are also part of the present invention.

[0100] Surprisingly, the compounds of the present invention show goodintegrin antagonistic activity. They are therefore suitable for thetreatment of diseases, especially as α₄β₁, and/or α₄β₇ and/or α₉β₁integrin antagonists and in the manufacture of a medicament for thetreatment or the prevention of a condition mediated by integrins and inparticular for the production of pharmaceutical compositions for theinhibition or the prevention of cell adhesion and cell-adhesion mediateddisorders. Examples are the treatment and the prophylaxis ofatherosclerosis, asthma, chronic obstructive pulmonary disease (COPD),allergies, diabetes, inflammatory bowel disease, multiple sclerosis,myocardial ischemia, rheumatoid arthritis, transplant rejection andother inflammatory, autoimmune and immune disorders.

[0101] The integrin antagonists of the invention are useful not only fortreatment of the physiological conditions discussed above, but are alsouseful in such activities as purification of integrins and testing foractivity.

[0102] For the treatment of the above-mentioned diseases, the compoundsaccording to the invention can exhibit non-systemic or systemicactivity, wherein the latter is preferred. To obtain systemic activitythe active compounds can be administered, among other things, orally orparenterally, wherein oral administration is preferred.

[0103] For parenteral administration, forms of administration to themucous membranes (i.e. buccal, lingual, sublingual, rectal, nasal,pulmonary, conjunctival or intravaginal) or into the interior of thebody are particularly suitable. Administration can be carried out byavoiding absorption (i.e. intracardiac, intra-arterial, intravenous,intraspinal or intralumbar administration) or by including absorption(i.e. intracutaneous, subcutaneous, percutaneous, intramuscular orintraperitoneal administration).

[0104] For the above purpose the active compounds can be administeredper se or in administration forms.

[0105] Suitable administration forms for oral administration are, interalia, normal and enteric-coated tablets, capsules, coated tablets,pills, granules, pellets, powders, solid and liquid aerosols, syrups,emulsions, suspensions and solutions. Suitable administration forms forparenteral administration are injection and infusion solutions.

[0106] The active compound can be present in the administration forms inconcentrations of from 0.001-100% by weight; preferably theconcentration of the active compound should be 0.5-90% by weight, i.e.quantities which are sufficient to allow the specified range of dosage.

[0107] The active compounds can be converted in the known manner intothe abovementioned administration forms using inert non-toxicpharmaceutically suitable auxiliaries, such as for example excipients,solvents, vehicles, emulsifiers and/or dispersants.

[0108] The following auxiliaries can be mentioned as examples: water,solid excipients such as ground natural or synthetic minerals (e.g.talcum or silicates), sugar (e.g. lactose), non-toxic organic solventssuch as paraffins, vegetable oils (e.g. sesame oil), alcohols (e.g.ethanol, glycerol), glycols (e.g. polyethylene glycol), emulsifyingagents, dispersants (e.g. polyvinylpyrrolidone) and lubricants (e.g.magnesium sulphate).

[0109] In the case of oral administration tablets can of course alsocontain additives such as sodium citrate as well as additives such asstarch, gelatin and the like. Flavour enhancers or colorants can also beadded to aqueous preparations for oral administration.

[0110] For the obtainment of effective results in the case of parenteraladministration it has generally proven advantageous to administerquantities of about 0.001 to 100 mg/kg, preferably about 0.01 to 1 mg/kgof body weight. In the case of oral administration the quantity is about0.01 to 100 mg/kg, preferably about 0.1 to 10 mg/kg of body weight.

[0111] It may nevertheless be necessary to use quantities other thanthose mentioned above, depending on the body weight concerned, themethod of administration, the individual response to the activecompound, the type of preparation and the time or interval ofadministration.

[0112] Pharmaceutically acceptable salts of the compounds of the presentinvention that contain an acidic moiety include addition salts formedwith organic or inorganic bases. The salt forming ion derived from suchbases can be metal ions, e.g., aluminum, alkali metal ions, such assodium of potassium, alkaline earth metal ions such as calcium ormagnesium, or an amine salt ion, of which a number are known for thispurpose. Examples include ammonium salts, arylalkylamines such asdibenzylamine and N,N-dibenzylethylenediamine, lower alkylamines such asmethylamine, t-butylamine, procaine, lower alkylpiperidines such asN-ethylpiperidine, cycloalkyl-amines such as cyclohexylamine ordicyclohexylamine, 1-adamantylamine, benzathine, or salts derived fromamino acids like arginine, lysine or the like. The physiologicallyacceptable salts such as the sodium or potassium salts and the aminoacid salts can be used medicinally as described below and are preferred.

[0113] Pharmaceutically acceptable salts of the compounds of the presentinvention that contain a basic moiety include addition salts formed withorganic or inorganic acids. The salt forming ion derived from such acidscan be halide ions or ions of natural or unnatural carboxylic orsulfonic acids, of which a number are known for this purpose. Examplesinclude chlorides, acetates, trifluoroacetates, tartrates, or saltsderived from amino acids like glycine or the like. The physiologicallyacceptable salts such as the chloride salts, the trifluoroacetic acidsalts and the amino acid salts can be used medicinally as describedbelow and are preferred.

[0114] These and other salts which are not necessarily physiologicallyacceptable are useful in isolating or purifying a product acceptable forthe purposes described below.

[0115] The compounds according to the invention can exist in differentstereoisomeric forms, which relate to each other in an enantiomeric way(image and mirror image) or in a diastereomeric way (image differentfrom mirror image). The invention relates to the enantiomers and thediastereomers as well as their mixtures. They can be separated accordingto customary methods.

[0116] General Compound Synthesis

[0117] The synthesis of compounds according to the general formula (I)can be illustrated by the following scheme 1:

[0118] By coupling of the carboxylic acid derivatives (II) with theamines (III), the amides (IV) can be obtained. Removal of the protectinggroup PG¹ followed by coupling with the amines (V) yields the amides(VI). If a protecting group PG₂ is used, the removal of the latteraffords carboxylic acids of type (VII).

[0119] In the above scheme the depicted ring in formulas (I), (II),(IV), and (VI) represents a cyclic moiety formed by R¹. AG stands forhydroxyl or a suitable activating group forming an activated carboxylicacid derivative. Activated carboxylic acids derivatives of this type areknown to the person skilled in the art and are described in detail instandard textbooks such as, for example in (i) Houben-Weyl, Methoden derorganischen Chemie [Methods of Organic Chemistry], Georg Thieme Verlag,Stuttgart or (ii) Comprehensive Organic Synthesis, Ed. B. M. Trost,Pergamon Press, Oxford, 1991. The carboxylic acid is preferablyactivated as, such as, for example, AG=1-hydroxy-1H-benzotriazol and acoupling agents such as, for example, dicyclohexylcarbodimid (DCC),1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide×HCl (EDCI),2-(7-aza-3-oxido-1H-1,2,3-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate. Other activated carboxylic acid derivatives suchas, for example symmetric anhydrides, mixed anhydrides, N-carboxyanhydrides, halides, or further activated esters e.g. succinyl orpentafluorophenyl esters may also be employed.

[0120] In the above scheme PG¹ and/or PG² stand for a suitableprotecting group of the carboxyl group or COOPG¹ and/or COOPG² stand forthe carboxylic group attached to a polymeric resin suitable for solidphase synthesis. Protecting groups of this type are known to the personskilled in the art and are described in detail in T. W. Greene, P. G.Wuts, Protective Groups in Organic Synthesis, 3^(rd) ed., John Wiley,New York, 1999. The carboxyl group is preferably esterified, PG¹ beingC₁₋₆-alkyl such as, for example, methyl, ethyl, propyl, isopropyl,butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, aC₃₋₇-cycloalkyl such as, for example, cyclopropyl, cyclopropylmethyl,cyclobutyl, cyclopentyl, cyclohexyl, an aryl such as, for example,phenyl, benzyl, tolyl or a substituted derivative thereof.

[0121] Step A

[0122] Formation of the amides (IV) can take place by reacting anactivated form of the respective carboxylic acid (II), such as a1-hydroxy-1H-benzotriazol ester with the desired amine (III) or anacceptable salt thereof. 1-Hydroxy-1H-benzotriazol ester of (II) can beprepared, for example, by the reaction of the 1-hydroxy-1H-benzbtriazolwith the carboxylic acids (II) in presence of an coupling agents suchas, for example, dicyclohexylcarbodiimid (DCC),1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide×HCl (EDCI),2-(7-aza-3-oxido-1H-1,2,3-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate. Further activated derivatives of the acids (II)such as other anhydrides, halides, esters e.g. succinyl orpentafluorophenyl esters or activated carboxylic acids obtained by thereaction with may also be employed.

[0123] For example, amides of type (IV) can be prepared as follows:

[0124] 1-Hydroxy-1H-Benzotriazol Ester Procedure:

[0125] A solution of carboxylic acid, 1-hydroxy-1H-benzotriazol (HOBt)and 1-ethyl-3-(3′-dimethylaminopropyl) carbodiimide×HCl (EDCI) in aninert solvent is stirred at r.t.. After addition of the amine and anon-nucleophilic base such as ethyldiisopropyl-amine or potassiumcarbonate stirring is continued at r.t. or elevated temperature. Afterevaporation, the residue was redissolved in ethyl acetate, washed withaqueous acid and base, dried and evaporated. If necessary the productwas purified by trituration or by flash-chromatography or used withoutfurther purification.

[0126] Compounds of general formula (II) are commercially available,known or can be prepared by customary methods starting from commerciallyavailable precursors.

[0127] Compounds of general formula (III) are commercially available,known or can be prepared by customary methods starting from knowncarboxylic acid derivatives.

[0128] Step B

[0129] The removal of the protecting group PG¹ can be performed eitherby an acid such as trifluoroacetic acid or an base such as potassiumhydroxide or lithium hydroxide, depending on the nature of PG¹.Reactions are carried out in aqueous, inert organic solvents such asalcohols e.g. methanol or ethanol, ethers e.g. tetrahydrofurane ordioxane or polar aprotic solvents e.g. dimethylformamide. If necessary,mixtures of the above solvents may be used.

[0130] Formation of the amides (VI) can take place by reacting therespective carboxylic acids (IV)-activated by a coupling agent such asDCC and HOBt; EDCI and HOBt or HATU—with the desired amines (V) or anacceptable salt thereof. Activated derivatives of the acids (IV) such asanhydrides, halides, and esters e.g. succinyl or pentafluorophenylesters may also be employed.

[0131] For example, amides (VI) can be prepared as follows:

[0132] A solution of carboxylic acid, 1-hydroxy-1H-benzotriazol (HOBt)and 1-ethyl-3-(3′-dimethylaminopropyl) carbodiimide×HCl (EDCI) in aninert solvent is stirred at r.t.. After addition of the amine and anon-nucleophilic base such as ethyldiisopropyl-amine or potassiumcarbonate stirring is continued at r.t. or elevated temperature. Afterevaporation, the residue was redissolved in ethyl acetate, washed withaqueous acid and base, dried and evaporated. If necessary the productwas purified by trituration or by flash-chromatography or used withoutfurther purification.

[0133] Compounds of general formula (V) are commercially available,known or can be prepared by customary methods starting from knowncarboxylic acid derivatives. Bisarylureas can be prepared by coupling ofan amino phenyl acetic acid derivative and a phenylisocyanate.

[0134] Step C

[0135] The removal of the protecting group PG² can be performed eitherby an acid such as trifluoroacetic acid or an base such as potassiumhydroxide or lithium hydroxide, depending on the nature of PG².Reactions are carried out in aqueous, inert organic solvents such asalcohols e.g. methanol or ethanol, ethers e.g. tetrahydrofurane ordioxane or polar aprotic solvents e.g. dimethylformamide. If necessary,mixtures of the above solvents may be used.

EXAMPLES

[0136] Abbreviations AcOH acetic acid Boc tert-butyloxycarbonyl DCCdicyclohexylcarbodiimid DCM dichloromethane DIPEA diisopropylethylamineEDCI 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimidexHCl eq. equivalentsEtOAc ethyl acetate FC flash chromatography GC gas chromatography HATU2-(7-aza-3-oxido-1H-1,2,3-benzotriazol-1-yl)-1,1,3,3- tetramethyluroniumhexafluorophosphate HOBt N-hydroxybenzotriazole monohydrate HPLC highperformance liquid chromatography ICAM-1 intracellular adhesion molecule1 IL-1 interleukin 1 LPS lipopolysaccharide MAdCAM-1 mucosal addressincell adhesion molecule 1 MeOH methanol MeCN acetonitril min. minutesM.p. melting point NE-κB nuclear factor κB NMR nuclear magneticresonance n.d. not determined PE light petroleum (b.p. 40-60° C.) r.t.room temperature R_(f) TLC: R_(f) value = distance spottraveled/distance solvent front traveled TFA trifluoroacetic acid THFtetrahydrofurane TLC thin layer chromatography TNF-α tumor necrosisfactor α t_(R) retention time determined by HPLC VCAM-1 vascular celladhesion molecule 1 VLA-4 very late antigen 4 (α₄β₁ integrin)

[0137] General Remarks

[0138] In the examples below, all quantitative data, if not statedotherwise, relate to percentages by weight.

[0139] Flash chromatography was carried out on silica gel 60, 40-63 μm.(E Merck, Darmstadt, Germany).

[0140] Thin layer chromatography was carried out, employing silica gel60 F₂₅₄ coated aluminum sheets (E. Merck, Darmstadt, Germany) with themobile phase indicated.

[0141] Melting points were determined in open capillaries and are notcorrected.

[0142] The mass determinations were carried out using the electron sprayionization (ESI) method employing loop injection or split injectionvia aHPLC system.

[0143] Precursor Synthesis

Example I N-(4-Aminophenyl)-N′-(2-methylphenyl)urea

[0144]

[0145] 2-Methylphenylisocyanate (24.6 g, 184.9 mmol) was added dropwiseat 0° C. to a solution of 1,4-diamino benzene (20.00 g, 184.9 mmol) in1000 mL EtOAc. After stirring for 2 h at r.t. the product was collectedby filtration (42.7 g, 177.0 mmol). M.p. >300° C.; TLC (PE/EtOAc 1/4)R_(f) 0.32; ¹H-NMR (400 MHz, D₆-DMSO) δ2.10 (s, 3H); 4.76 (s, 2H); 6.59(mc, 2H); 6.89 (mc, 1H); 7.07-7.15 (m, 4H); 7.73 (s, 1H); 7.85 (mc, 2H);8.50 (s, 1H).

Example II tert-Butyl4-({[(2-methylphenyl)amino]carbonyl}amino)benzyl-carbamate

[0146]

[0147] 2-Methylphenylisocyanate (7.57 g, 59.83 mmol) was added dropwiseat 0° C. to a solution of (4-amino-benzyl)-carbamic acid tert-butylester (13.30 g, 59.83 mmol, prepared analoguous to: Moloney, Gerard P.;Martin, Graeme R.; Mathews, Neil; Milne, Aynsley; Hobbs, Heather; et al.J Med. Chem. 1999, 42, 2504-2526) in 120 mL DCM. The reaction was heatedunder reflux for 16 h, cooled to r.t. and the precipitated product wascollected by filtration and dried in vacuum (19.20 g, 54.00 mmol). M.p.200-202° C.; TLC (PE/EtOAc 1/1) R_(f) 0.65; ¹H NMR (400 MHz, D₆-DMSO)δ1.39 (s, 9H); 2.24 (s, 3H); 4.06 (d, J=6 Hz, 2H); 6.93 (mc, 1H);7.12-7.17 (m, 4); 7.32 (mc, 1H); 7.40 (mc, 2H); 7.85 (mc, 1H); 7.90 (s,1H); 8.98 (s, 1H).

Example III N-[4-(Aminomethyl)phenyl]-N′-(2-methylphenyl)urea

[0148]

[0149] To a solution of tert-butyl4-({[(2-methylphenyl)amino]carbonyl}amino)benzylcarbamate (2.00 g, 5.63mmol) in CH₂Cl₂ (120 mL) TFA (36 mL) was added at 0° C. and stirred for2 h at r.t.. The reaction mixture was evaporated and the product wascollected (2.72 g, TFA salt). M.p. 142-143° C.; TLC (PE/EtOAc 3/2) R_(f)0.14; ¹H NMR (400 MHz, D₆-DMSO) δ2.24 (s, 3H); 3.97 (q, J=5 Hz, 2H);6.96 (mc, 1H); 7.13-7.19 (m, 2); 7.36 (mc, 2H); 7.51 (mc, 2H); 7.81 (mc,2H); 8.06 (s, 1H); 8.08 (s, 3H); 9.23 (s, 1H).

[0150] Compound Synthesis

[0151] Step A:

Example IV tert-Butyl4-({[1-(3,4-dimethoxyphenyl)-3-ethoxy-3-oxopropyl]-amino}carbonylbenzoate

[0152]

[0153] 4-(tert-Butoxycarbonyl)benzoic acid (42 mg, 0.19 mmol) wasdissolved in MeCN, HOBt (28 mg, 0.21 mmol), EDCI (41 mg, 0.21 mmol),DIPEA (50 μL, 0.29 mmol), and ethyl3-amino-3-(3,4-dimethoxyphenyl)propanoate*HCl (56 mg, 0.21 mmol) wereadded at r.t. After stirring for 24 h, the solvent was evaporated andthe residue was dissolved in EtOAc (200 mL), washed with 10% aqueouscitric acid (50 mL), sat. aqueous soda (50 mL) and dried (NaSO₄). Thesolvent was evaporated in vacuum and the product was collected (47 mg,0.10 mmol, 53%) as a colorless solid. M.p. 108° C.; TLC(cyclohexane/EtOAc 7/3) R_(f) 0.17; 1H NMR (400 MHz, CDCl₃) δ1.20 (t,J=7.2 Hz, 3H); 1.26 (mc, 1H); 1.60 (s, 9H); 2.96 (mc, 2H); 3.86 (s, 3H);(s, 3H); 4.12 (q, J=7.1 Hz, 2H); 5.37 (mc, 1H); 6.83 (m, 1H); 6.89 (mc,1H); 7.55 (mc, 1H); 7.85 (mc, 2H); 8.05 (mc, 2H). TABLE 1 The followingexamples were prepared according to the general procedure Ex.- NoStructure Name M.p. (° C.) IV

tert-Butyl 4-({[1-(3,4-di- methoxyphenyl)-3-ethoxy-3-oxopropyl]amino}carbonyl) benzoate 108 V

tert-Butyl 4-[({[1-(ethoxy- carbonyl)cyclohexyl]methyl}-amino)carbonyl]benzoate 104 VI

tert-Butyl 4-({[3-ethoxy-1-(3- methoxyphenyl)-3-oxo-propyl]amino}carbonyl)- benzoate 102

[0154] Step B:

Example VIItert-Butyl-4-{[(1-(3,4-dimethoxyphenyl)-3-{[4-({[(2-methylphenyl)-amino]carbonyl}amino)phenyl]amino}-3-oxopropyl)amino]carbonyl}benzoate

[0155]

[0156]tert-Butyl-4-({[1-(3,4-dimethoxyphenyl)-3-ethoxy-3-oxopropyl]amino}carbonyl)benzoate(40 mg, 0.09 mmol) was dissolved in THF/water (v/v 1/1) and lithiumhydroxide (2.3 mg, 0.1 mmol) was added at r.t and the reaction mixturewas stirred for 24 h. The solvent was removed under vacuum and thelithium salt of3-{[4-(tert-butoxycarbonyl)benzoyl]amino}-3-(3,4-dimethoxyphenyl)propanoicacid was isolated (37 mg, 0.09 mmol). The latter compound (18 mg, 0.04mmol) was dissolved in MeCN (1 mL), HOBt (5.5 mg, 0.05 mmol), EDCI (8.8mg, 0.05 mmol), and N-(4-aminophenyl)-N′-(2-methylphenyl) urea (10.0 mg,0.05 mmol) were added. After stirring for 24 h, the solvent wasevaporated and the residue was dissolved in EtOAc (200 mL), washed with10% aqueous citric acid (50 mL), sat. aqueous soda (50 mL) and dried(NaSO₄). The solvent was evaporated in vacuum and the product wascollected (27 mg, 0.10 mmol, 98%) as a colorless solid. M.p. 210-215°C.; TLC (DCM/MeOH 9/1) R_(f) 0.21; 1H NMR (400 MHz, D₆-DMSO) δ1.56 (s,9H); 2.23 (s, 3H); 2.86 (mc, 1H); 2.93 (mc, 1H); 3.71 (s, 3H); 3.74 (s,3H); 5.44 (mc, 1H); 6.88 (mc, 1H); 7.06 (mc, 1H); 7.14 (mc, 1H); 7.36(mc, 2H); 7.45 (mc, 2H); 7.67 (mc, 1H); 7.81 (mc, 1H); 7.90-8.02 (m,6H); 9.05 (s, 1H); 9.10 (s, 2H); 9.94 (s, 1H). TABLE 2 The followingexamples were prepared according to the general procedure Ex.- No.Structure Name M.p. (° C.) VII

tert-Butyl 4-{[(1-(3,4- dimethoxyphenyl)-3-{[4- ({[(2-methylphenyl)amino]carbonyl}amino) phenyl]-amino}-3-oxopropyl)-amino]carbonyl}benzoate 210-215 VIII

tert-Butyl 4-[({[1- ({[4-({[(2-methylphenyl) amino]carbonyl}amino)benzyl]amino}carbonyl)cyclohexyl]methyl}-amino) carbonyl]benzoate187-189 IX

tert-Butyl-4-{[(1-(3,4- dimethoxyphenyl)-3-{[4- ({[(2-methylphenyl)amino]carbonyl}amino) benzyl]amino}-3-oxopropyl)amino]carbonyl}-benzoate 223-226 X

tert-Butyl 4-{[(1-(3,4- dimethoxyphenyl)-3-{[4- ({[(2-methylphenyl)amino]carbonyl}amino) phenyl]amino}-3-oxopropyl)amino]carbonyl}-benzoate n.d.

[0157] Step C

Example 14-{[(1-(3,4-Dimethoxyphenyl)-3-{[4-({[(2-methylphenyl)amino]carbonyl}amino)phenyl]amino}-3-oxopropyl)amino]carbonyl}benzoicacid

[0158]

[0159]tert-Butyl-4-{[(1-(3,4-dimethoxyphenyl)-3-{[4-({[(2-methylphenyl)amino]carbonyl}amino)phenyl]amino}-3-oxopropyl)amino]carbonyl}benzoate (30 mg, 0.05 mmol) wasdissolved in DCM (10 mL) and TFA (0.18 mL, 2.30 mmol) was added at 0° C.and the reaction mixture was stirred at r.t. for 24 h. The solvent wasremoved in vacuum and the product was isolated (25 mg, 91%). M.p. 166°C. ESI-MS: 598[M+H]⁺. TABLE 3 The following examples were preparedaccording to the general procedure No Structure Name M.p. (° C.) 2

4-[({[1-({[4-({[(2-methylphenyl)amino]carbonyl}-amino)benzyl]amino}carbonyl)cyclohexyl]methyl}- amino)carbonyl]benzoic acid n.d. 3

4-{[(1-(3,4-dimethoxy- phenyl)-3-{[4-({[(2-methyl-phenyl)amino]carbonyl}amino)benzyl]amino}-3-oxopropyl)amino]carbonyl}benzoic acid 214 4

4-{[(1-(3-methoxy- phenyl)-3-{[4-({[(2-methylphenyl)-amino]carbonyl}amino)phenyl]amino}-3-oxopropyl)amino]carbonyl}benzoic acid 275-280

[0160] In Vitro Assay: Adhesion of Ramos Cells to Immobilized VCAM-1(Domains 1-3)

[0161] Preparation of VCAM-1 (extracellular domains 1-3)

[0162] Complementary DNA (cDNA) encoding 7-domain form of VCAM-1(GenBank accession #M60335) was obtained using Rapid-ScreenTM cDNAlibrary panels (OriGene Technologies, Inc) at Takara Gene AnalysisCenter (Shiga, Japan). The primers used were 5′-CCA AGG CAG AGT ACG CAAAC-3′ (sense) and 5′-TGG CAG GTA TTA TTA AGG AG-3′ (antisense). PCRamplification of the 3-domain VCAM-1 cDNA was perform using Pfu DNApolymerase (Stratagene) with the following sets of primers: (U-VCAMd1-3)5′-CCA TAT GGT ACC TGA TCA ATT TAA AAT CGA GAC CAC CCC AGA A-3′;(L-VCAMd1-3) 5′-CCA TAT AGC AAT CCT AGG TCC AGG GGA GAT CTC AAC AGTAAA-3′. PCR cycle was 94° C. for 45 sec, 55° C. for 45 sec, 72° C. for 2min, repeating 15 cycles. After the purification of the PCR product, thefragment was digested with KpnI-AvrII. The digested fragment was ligatedinto pBluescript IISK(-) (Strategene), which was linearized by digestingwith KpnI-XhoI. The ligation was followed by transformation to a Dam/Dcmmethylase-free E. coli strain SCS110 (Strategene) to create the donorplasmid pHH7. To direct VCAM-1 molecule into the insect cell secretorypathway, the VCAM-1 coding sequence was fused to signal peptide sequenceof honeybee melittin. The resulting melittin-VCAM fusion was placed incorrect orientation to the baculovirus polyhedrin promoter. Baculovirustransfer vector containing first 3-domain form VCAM-1 (pH10) wasconstructed by ligation of 0.9 kb fragment from AvrIIenow/BclI digestsof pH7 into SalI/Klenow/BamHI digests of pMelBacB (Invitrogen).Recombinant baculovirus was generated by using Bac-N-Blue™ Transfectionkit (Invitrogen) according to the manufacture's instruction. Therecombinant virus was amplified by infection to High-Five™ insect cellsfor 5-6 days, and virus titer was determined by plaque assay.

[0163] High-Five™ insect cells were pelleted in a 225 ml conical tube bycentrifugation at 1000 rpm for 5 min. After discarding the supernatant,the pellet was resuspended in 1.5×10⁹ pfu (MOI=5) of high-titer virussolution, followed by incubation for 1.5 hours at room temperature. Thecells were pelleted again and washed once in fresh Express Five™ serumfree medium. The cells were pelleted again and finally, resuspended in200 ml of fresh Express Five™ medium, transferred to a 1,000 ml shakerflask, and incubated in a shaker at 27° C., 130 rpm, for 48 hours beforethe culture supernatant was collected. The purification of 3-domain formof VCAM-1 from the culture supernatant was performed by one-step anionexchange chromatography. Protein concentration was determined by usingCoomassie protein assay reagent (Pierce) according to the manufacture'sinstruction.

[0164] Preparation of VCAM-1 Coated Microtiter Plates

[0165] Recombinant human VCAM-1 (extracellular domains 1-3) wasdissolved at 1.0 μg/ml in PBS. Each well of the microtiter plates (NalgeNunc International, Fluoronunc Cert, 437958) was coated with 100 μl ofsubstrate or for background control with buffer alone for 15 hours at 4C. After discarding the substrate solution, the wells were blocked using150 μl per well of block solution (Kirkegaard Perry Laboratories,50-61-01) for 90 minutes. The plate was washed with wash buffercontaining 24 mM Tris-HCl (pH 7.4), 137 mM NaCl, 27 mM KCl and 2 mMMnCl₂ just before addition of the assay.

[0166] In Vitro Assay Using Ramos Cells

[0167] Preparation of Fluorescence Labeled Ramos Cells:

[0168] Ramos cells (American Type Culture Collection, Clone CRL-1596)were cultured in RPMI 1640 medium (Nikken Bio Medical Laboratory,CM1101) supplemented with 10% fetal bovine serum (Hyclone, A-1119-L),100 U/ml penicilin (Gibco BRL, 15140-122) and 100 μg/ml streptomycin(Gibco BRL, 15140-122) in a humidified incubator at 37° C. with 5% CO₂.

[0169] Ramos cells were incubated with phosphate balanced solution (PBS,Nissui, 05913) containing 25 μM of 5(-and -6)-carboxyfluoresceindiacetate, succinimidyle ester (CFSE, Dojindo Laboratories, 345-06441)for 20 min at room temperature while gently swirling every 5 min. Aftercentrifugation at 1000 rpm for 5 min, the cell pellet was resuspendedwith adhesion assay buffer at a cell density of 4×10⁶ cells/ml. Theadhesion assay buffer was composed of 24 mM Tris-HCl (pH 7.4), 137 mMNaCl, 27 mM KCl, 4 mM glucose, 0.1% bovine serum albumin (BSA, Sigma,A9647) and 2 mM MnCl₂.

[0170] Assay Procedure (Ramos Cells)

[0171] The assay solution containing each test compounds or 5 μg/mlanti-CD49d monoclonal antibody (Immunotech, 0764) was transferred to theVCAM-1 coated plates. The final concentration of each test compounds was5 μM, 10 μM or various concentrations ranging from 0.0001 μM to 10 μMusing a standard 5-point serial dilution. The assay solution containingthe labeled Ramos cells was transferred to the VCAM-1 coated plates at acell density of 2×10⁵ cells per well and incubated for 1 hour at 37 C.The non-adherent cells were removed by washing the plates 3 times withwash buffer. The adherent cells were broken by addition of 1% TritonX-100 (Nacalai Tesque, 355-01). Released CFSC was quantifiedfluorescence measurement in a fluorometer (Wallac, ARVO 1420 multilabelcounter).

[0172] The adhesion of Ramos cells to VCAM-1 was analyzed by percentbinding calculated by the formula: 100×(FTS−FBG)/(FTB−FBG)=% binding,where FTB is the total fluorescent intensity from VCAM-1 coated wellswithout test compound; FBG is the fluorescent intensity from wells withanti-CD49d monoclonal antibody and FTS is the fluorescent intensity fromwells containing the test compound of this invention.

[0173] In Vitro Activity

[0174] In the Ramos VCAM-1 assay the observed IC₅₀ value ranges areindicated in Table 4.

C>10 μM≧B>1 μM≧A TABLE 4 No IC₅₀ 1 A 2 C 3 C 4 A

1. Compounds of the general formula (I),

wherein R¹ represents a 4- to 9-membered saturated, unsaturated oraromatic cyclic residue, which can contain 0 to 3 heteroatoms selectedindependently from the group N, S and O, and wherein R¹ is substitutedby —R¹⁻¹—Z, wherein R¹⁻¹ represents a bond, —O—, —S—, NR¹⁻², C₁-C₁₀alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₆ or C₁₀ aryl, C₃-C₇ cycloalkylor a 4-9-membered saturated or unsaturated heterocyclic residuecontaining up to 3 heteroatoms selected from the group oxygen, nitrogenor sulfur, wherein R¹⁻¹ can optionally be substituted by 1 to 2substituents selected from the group R¹⁻³, wherein R¹⁻² can optionallybe hydrogen, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl or C₂-C₁₀ alkynyl, and whereinR¹⁻³ represents hydrogen, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl,C₆ or C₁₀ aryl, C₃-C₇ cycloalkyl or a 4-9-membered saturated orunsaturated heterocyclic residue containing up to 3 heteroatoms selectedfrom the group oxygen, nitrogen or sulfur, Z represents —C(O)OR^(Z-1),—C(O)NR^(Z-2)R^(Z-3), —SO₂NR^(Z-2)R^(Z-3), —SO(OR^(Z-1)),—SO₂(OR^(Z-1)), —P(O)R^(Z-1)(OR^(Z-3)) or —PO(OR^(Z-1))(OR^(Z-3)),wherein R^(Z-2) is hydrogen, C₁-C₄ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,C₃-C₆ cycloalkyl, C₆ or C₁₀ aryl, —C(O)R^(Z-4) or —SO₂R^(Z-4), whereinR^(Z-4) is C₁-C₄ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl,C₆ or C₁₀ aryl, R^(Z-1) and R^(Z-3) are independently selected from thegroup hydrogen, C₁-C₄ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆cycloalkyl, C₆ or C₁₀ aryl or benzyl, wherein R^(Z-1) and R^(Z-3) canoptionally be substituted by 1 to 3 substituents selected from the groupC₁-C₄ alkyl, C₁-C₄ alkyloxy, halogen, nitro, cyano, and wherein R¹ canoptionally be substituted by 0 to 2 substituents R¹⁻⁴, halogen, nitro,amino, cyano and oxo, wherein R¹⁻⁴ is selected from the group C₁-C₄alkyl, C₁-C₄ alkyloxy, phenyl, phenoxy, phenylamino, C₃-C₆ cycloalkyl,R² represents hydrogen, C₁-C₁₀ alkyl, C₂-C10 alkenyl, C₂-C₁₀ alkynyl, C₆or C₁₀ aryl or C₃-C₇ cycloalkyl, wherein R² can optionally besubstituted by 1 to 3 radicals independently selected from the groupC₁-C₄ alkyl, trifluormethyl, trifluormethoxy, halogen, cyano, nitro oroxo, R³ represents hydrogen, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀alkynyl, C₆ or C₁₀ aryl, C₃-C₇ cycloalkyl or a 4-9-membered saturated orunsaturated heterocyclic residue containing up to 2 heteroatoms selectedfrom the group oxygen, nitrogen or sulfur, wherein R³ can optionally besubstituted by 1 to 3 radicals R³⁻¹, wherein R³⁻¹ represents C₁-C₄alkyl, trifluormethyl, trifluormethoxy, —OR³⁻², —SR³⁻², NR³⁻³R³⁻⁴,—C(O)R³⁻², S(O)R³⁻², —SO₂R³⁻², —OC(O)R³⁻², —C(O)NR³⁻³R³⁻⁴,—NR³⁻²C(O)R³⁻³, —SO₂NR³⁻³R³⁻⁴, NR³⁻²SO₂R³⁻³, —NR³⁻²C(O)NR³⁻³R³⁻⁴,—NR³⁻²C(O)OR³⁻³, —OC(O)NR³⁻³R³⁻⁴, —CO₂R³⁻⁵, halogen, cyano, nitro oroxo, wherein R³⁻² represents hydrogen, C₁-C₄ alkyl, C₃-C₆ cycloalkyl, C₆or C₁₀ aryl, wherein R³⁻³ and R³⁻⁴ are independently selected from thegroup hydrogen, C₁-C₄ alkyl, C₃-C₆ cycloalkyl, C₆ or C₁₀ aryl or benzyl,and wherein R³⁻⁵ represents C₁-C₄ alkyl, C₃-C₆ cycloalkyl, C₆ or C₁₀aryl, R⁴ represents hydrogen, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀alkynyl, C₆ or C₁₀ aryl, C₃-C₇ cycloalkyl or a 4-9-membered saturated orunsaturated heterocyclic residue containing up to 2 heteroatoms selectedfrom the group oxygen, nitrogen or sulfur, wherein R⁴ can optionally besubstituted by 1 to 3 radicals R⁴⁻¹, wherein R⁴⁻¹ represents C₁-C₄alkyl, trifluormethyl, trifluormethoxy, —OR⁴⁻², —SR⁴⁻², NR⁴⁻³R⁴⁻⁴,—C(O)R⁴⁻², S(O)R⁴⁻², —SO₂R⁴⁻², —OC(O)R⁴⁻², —C(O)NR⁴⁻³R⁴⁻⁴,—NR⁴⁻²C(O)R⁴⁻³, —SO₂NR⁴⁻³R⁴⁻⁴, NR⁴⁻²SO₂R⁴⁻³, —NR⁴⁻²C(O)NR⁴⁻³R⁴⁻⁴,—NR⁴⁻²C(O)OR⁴⁻³, —OC(O)NR⁴⁻³R⁴⁻⁴, —CO₂R⁴⁻⁵, halogen, cyano, nitro oroxo, wherein R⁴⁻² represents hydrogen, C₁-C₄ alkyl, C₃-C₆ cycloalkyl, C₆or C₁₀ aryl, wherein R⁴⁻³ and R⁴⁻⁴ are independently selected from thegroup hydrogen, C₁-C₄ alkyl, C₃-C₆ cycloalkyl, C₆ or C₁₀ aryl or benzyl,and wherein R⁴⁻⁵ represents C₁-C₄ alkyl, C₃-C₆ cycloalkyl, C₆ or C₁₀aryl or R³ and R⁴ together with the carbon atom to which they areattached form a 4-7-membered saturated or unsaturated ring containing upto 2 heteroatoms selected from the group oxygen, nitrogen or sulfur,which can optionally be substituted by 1 to 2 substituents selected fromthe group C₁-C₄ alkyl, phenyl, benzyl, C₃-C₇ cycloalkyl, C₁-C₄ alkyloxy,halogen, nitro, cyano, oxo and which can be fused with a 3-7 memberedhomocyclic or heterocyclic, saturated, unsaturated or aromatic ring, R⁵represents hydrogen, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₆ orC₁₀ aryl or C₃-C₇ cycloalkyl, wherein R⁵ can optionally up tothreefoldedly be substituted by C₁-C₄ alkyl, trifluormethyl,trifluormethoxy, halogen, cyano, nitro or oxo, R⁶ represents phenyl or a5- to 6-membered aromatic heterocyclic residue containing up to 3heteroatoms independently selected from the group oxygen, nitrogen orsulfur, which is substituted by —NR⁶⁻²C(O)NR⁶⁻³R⁶⁻⁴ or—NR⁶⁻²C(S)NR⁶⁻³R⁶⁻⁴ and can furthermore optionally be substituted byhalogen, wherein R⁶⁻² and R⁶⁻³ are independently selected from the grouphydrogen or C₁-C₄ alkyl, or together form a group

and wherein R⁶⁻⁴ represents phenyl, wherein R⁶⁻⁴ can optionally besubstituted by 1-2 substituents selected from the group C₁-C₄ alkyl,C₁-C₄ alkyloxy, halogen, nitro, triluoromethyl, trifluoromethoxy orcyano, R⁷ represents hydrogen, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀alkynyl, C₆ or C₁₀ aryl, C₃-C₇ cycloalkyl or a 4-9-membered saturated orunsaturated heterocyclic residue containing up to 2 heteroatoms selectedfrom the group oxygen, nitrogen or sulfur, wherein R⁷ can optionally besubstituted by 1 to 3 radicals R⁷⁻¹, wherein R⁷⁻¹ represents C₁-C₄alkyl, trifluormethyl, trifluormethoxy, —OR⁷⁻², —SR⁷⁻², NR⁷⁻³R⁷⁻⁴,—C(O)R⁷⁻², S(O)R⁷⁻², —SO₂R⁷⁻², —OC(O)R⁷⁻², —C(O)NR⁷⁻³R⁷⁻⁴,—NR⁷⁻²C(O)R⁷⁻³, —SO₂NR⁷⁻³R⁷⁻⁴, NR⁷⁻²SO₂R⁷⁻³, —NR⁷⁻²C(O)NR⁷⁻³R⁷⁻⁴,—NR⁷⁻²C(O)OR⁷⁻³, —OC(O)NR⁷⁻³R⁷⁻⁴, —CO₂R⁷⁻⁵, halogen, cyano, nitro oroxo, wherein R⁷⁻² represents hydrogen, C₁-C₄ alkyl, C₃-C₆ cycloalkyl, C₆or C₁₀ aryl, wherein R⁷⁻³ and R⁷⁻⁴ are independently selected from thegroup hydrogen, C₁-C₄ alkyl, C₃-C₆ cycloalkyl, C₆ or C₁₀ aryl or benzyl,and wherein R⁷⁻⁵ represents C₁-C₄ alkyl, C₃-C₆ cycloalkyl, C₆ or C₁₀aryl R⁸ represents hydrogen, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀alkynyl, C₆ or C₁₀ aryl, C₃-C₇ cycloalkyl or a 4-9-membered saturated orunsaturated heterocyclic residue containing up to 2 heteroatoms selectedfrom the group oxygen, nitrogen or sulfur, wherein R⁸ can optionally besubstituted by 1 to 3 radicals R⁸⁻¹, wherein R⁸⁻¹ represents C₁-C₄alkyl, trifluormethyl, trifluormethoxy, —OR⁸⁻², —SR⁸⁻², NR⁸⁻²R⁸⁻⁴,—C(O)R⁸⁻², S(O)R⁸⁻², —SO₂R⁸⁻², —OC(O)R⁸⁻², —C(O)R⁸⁻³R⁸⁻⁴,—NR⁸⁻²C(O)R⁸⁻³, —SO₂NR⁸⁻³R⁸⁻⁴, NR⁸⁻²SO₂R⁸⁻³, —NR⁸⁻²C(O)NR⁸⁻³R⁸⁻⁴,—NR⁸⁻²C(O)R⁸⁻³, —OC(O)NR⁸⁻³R⁸⁻⁴, —CO₂R⁸⁻⁵, halogen, cyano, nitro or oxo,wherein R⁸⁻² represents hydrogen, C₁-C₄ alkyl, C₃-C₆ cycloalkyl, C₆ orC₁₀ aryl, wherein R⁸⁻³ and R⁸⁻⁴ are independently selected from thegroup hydrogen, C₁-C₄ alkyl, C₃-C₆ cycloalkyl, C₆ or C₁₀ aryl or benzyl,and wherein R⁸⁻⁵ represents C₁-C₄ alkyl, C₃-C₆ cycloalkyl, C₆ or C₁₀aryl or R⁷ and R⁸ together form a 4-7-membered saturated or unsaturatedring containing up to 2 heteroatoms selected from the group oxygen,nitrogen or sulfur, which can optionally be substituted by 1 to 2substituents selected from the group C₁-C₄ alkyl, phenyl, benzyl, C₃-C₇cycloalkyl, C₁-C₄ alkyloxy, halogen, nitro, cyano, oxo and which can befused with a 3-7 membered homocyclic or heterocyclic, saturated,unsaturated or aromatic ring, X represents bond or(—CR^(X-1)R^(X-2)—)_(n), wherein R^(X-1) and R^(X-2) can beindependently selected from the group hydrogen, C₁-C₄ alkyl, C₂-C₄alkenyl, C₂-C₄ alkynyl, wherein R^(X-1) and R^(X-2) can optionallyindependently be substituted by 1 to 2 substituents selected from thegroup C₁-C₄ alkyl, phenyl, benzyl, C₃-C₇ cycloalkyl, C₁-C₄ alkyloxy,halogen, nitro, cyano, oxo, and wherein n is an integer 0 or 1 andpharmaceutically acceptable salts thereof.
 2. Compounds of generalformula (I) according to claim 1, wherein R¹ represents a phenyl ring.3. Compounds according to claim 1 or 2, wherein R¹⁻¹ represents a bondand Z represents COOR^(Z-1), wherein R^(Z-1) has the meaning indicatedabove.
 4. Compounds according to claim 1, 2 or 3, wherein R⁶ representsphenyl, which is substituted by —NHC(O)NHR⁶⁻⁴, wherein R⁶⁻⁴ issubstituted with methyl or trifluoromethoxy.
 5. Compounds according toany one of claims 1 to 4, wherein R^(X-1) and R^(X-2) representhydrogen.
 6. Compounds according to any one of claims 1 to 5, wherein R³and R⁴ together form a 6-membered homocycle.
 7. Compounds according toany one of claims 1 to 6, wherein R³, R⁴ and R⁷ represent hydrogen andR⁸ represents a 3-methoxyphenylradical or a 3,4-dimethoxyphenylradical.8. Compounds of general formula (I) according to claim 2, characterizedin that R¹ is a 1,4-substituted phenyl ring.
 9. A process forpreparation of compounds of general formula (I) according to any one ofclaims 1 to 7, which comprises reaction of carboxylic acids of generalformula (I′)

or activated derivatives thereof, with compounds of the general formula(I″) R⁶—X—NR⁵H  (I″) in inert solvents.
 10. Compounds according to anyone of claims 1 to 7 for the treatment of diseases.
 11. The use of acompound according to any one of claims 1 to 7 in the manufacture of amedicament for the treatment or the prevention of a condition mediatedby integrins.
 12. The use of a compound according to any one of claims 1to 7 in the manufacture of a medicament for the treatment or theprevention of atherosclerosis, asthma, chronic obstructive pulmonarydisease (COPD), allergies, diabetes, inflammatory bowel disease,multiple sclerosis, myocardial ischemia, rheumatoid arthritis,transplant rejection and other inflammatory, autoimmune and immunedisorders.
 13. Pharmaceutical composition, comprising compoundsaccording to any one of claims 1 to 7 and a pharmaceutically acceptablecarrier.
 14. Compounds of general formula (I′),

wherein R¹, R², R³, R⁴, R⁷ and R⁸ have the abovementioned meaning andPG₁ represents a protecting group for the carboxyl group.